Magnesium is an essential mineral in vertebrates and involved in many biochemical and physiological functions. Magnesium homeostasis is regulated between the overall requirements of the body, and the renal and intestinal reabsorption and excretion. Despite extensive study, the molecular mechanisms of magnesium regulation are poorly understood. Since 1997, we have studied an autosomal recessive disorder, familial hypomagnesemia with secondary hypocalcemia (HSH). Patients affected with this disease show severe hypomagnesemia and hypocalcemia, which leads to tetany and seizures, which can be lethal if left untreated. The disorder has been thought to be caused by a defect in the intestinal absorption of magnesium, rather than by abnormal renal loss of magnesium because HSH patients have low to normal levels of magnesium in their urine, prior to treatment. High-dose magnesium supplementation, which is needed as a life-long treatment, can overcome the apparent defect in magnesium absorption and in serum concentrations of calcium. We initially mapped the gene locus to chromosome 9q in three large inbred kindreds from Israel. The HSH locus was subsequently narrowed to approximately 2 Mb, flanked by D9S1115 and D9S 175. We recently demonstrated that mutations in TRPM6, a candidate gene within the narrowed interval, caused HSH. TRPM6 is highly homologous to a bifunctional protein, TRPM7, which can act as an ion channel for divalent cations and as a serine/threonine protein kinase. We also observed that patients carrying mutations in TRPM6 have abnormal renal magnesium excretion. These findings indicate that TRPM6 plays an important role in magnesium homeostasis in the kidney, as well as in the intestine. We would like to extend these studies to determine the precise locations of TRPM6 expression in the kidney, and to compare it with expression of TRPM7. TRPM6 is selectively expressed in colon and in kidney, whereas TRPM7 is widely expressed, and also expressed in the kidney. We will develop specific, non-cross-reacting antibodies to the two proteins for immunolocalization and Western studies. We will examine the possibility of co-localization of the two proteins. TRPM6 and TRPM7 expression will be examined in mice made hypomagnesemic with magnesium deprivation diets to test if expression is regulated in hypomagnesemia and hypocalcemia. The expression of the two TRP channels will also be examined in mice on calcium deprivation diet.